p-i snake venom metalloproteinase is able to activate the

P-I Snake Venom Metalloproteinase Is Able to Activatethe Complement System by Direct Cleavage of CentralComponents of the CascadeGiselle Pidde-Queiroz1, Fabio Carlos Magnoli1, Fernanda C. V. Portaro1, Solange M. T. Serrano2, Aline

Soriano Lopes2, Adriana Franco Paes Leme3, Carmen W. van den Berg4, Denise V. Tambourgi1*

1 Laboratorio de Imunoquımica, Instituto Butantan, Sao Paulo, Brazil, 2 Laboratorio de Toxinologia Aplicada, Instituto Butantan, Sao Paulo, Brazil, 3 Laboratorio Nacional

de Biociencias, Centro Nacional de Pesquisa em Energia e Materiais, Campinas, Sao Paulo, Brazil, 4 Institute of Molecular and Experimental Medicine, School of Medicine,

Cardiff University, Cardiff, United Kingdom

Abstract

Background: Snake Venom Metalloproteinases (SVMPs) are amongst the key enzymes that contribute to the high toxicity ofsnake venom. We have recently shown that snake venoms from the Bothrops genus activate the Complement system (C) bypromoting direct cleavage of C-components and generating anaphylatoxins, thereby contributing to the pathology andspread of the venom. The aim of the present study was to isolate and characterize the C-activating protease from Bothropspirajai venom.

Results: Using two gel-filtration chromatography steps, a metalloproteinase of 23 kDa that activates Complement wasisolated from Bothrops pirajai venom. The mass spectrometric identification of this protein, named here as C-SVMP, revealedpeptides that matched sequences from the P-I class of SVMPs. C-SVMP activated the alternative, classical and lectin C-pathways by cleaving the a-chain of C3, C4 and C5, thereby generating anaphylatoxins C3a, C4a and C5a. In vivo, C-SVMPinduced consumption of murine complement components, most likely by activation of the pathways and/or by directcleavage of C3, leading to a reduction of serum lytic activity.

Conclusion: We show here that a P-I metalloproteinase from Bothrops pirajai snake venom activated the Complementsystem by direct cleavage of the central C-components, i.e., C3, C4 and C5, thereby generating biologically active fragments,such as anaphylatoxins, and by cleaving the C1-Inhibitor, which may affect Complement activation control. These resultssuggest that direct complement activation by SVMPs may play a role in the progression of symptoms that followenvenomation.

Citation: Pidde-Queiroz G, Magnoli FC, Portaro FCV, Serrano SMT, Lopes AS, et al. (2013) P-I Snake Venom Metalloproteinase Is Able to Activate the ComplementSystem by Direct Cleavage of Central Components of the Cascade. PLoS Negl Trop Dis 7(10): e2519. doi:10.1371/journal.pntd.0002519

Editor: Jean-Philippe Chippaux, Institut de Recherche pour le Developpement, Benin

Received July 28, 2013; Accepted September 21, 2013; Published October 31, 2013

Copyright: � 2013 Pidde-Queiroz et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This research was supported by grants from Instituto Nacional de Ciencia e Tecnologia em Toxinas (INCTTOX), Fundacao de Amparo a Pesquisa doEstado de Sao Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Cientıfico e Tecnologico (CNPq). The funders had no role in study design, datacollection and analysis, decision to publish, or preparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: [email protected]

Introduction

The venom of Viperidae snakes is a bioactive mixture of

proteases, peptides, and non-enzymatic proteins that interact with

multiple components of the body affecting the hemostatic system.

The genus Bothrops inflicts the vast majority of snakebites in

Central and South America and is responsible for 90% of snake

envenomations in Brazil [1–3].

Envenomations are characterized by prominent local effects,

including edema, hemorrhage and necrosis, which can lead to

permanent disability. Systemic manifestations, such as hemor-

rhage, coagulopathy, shock and acute renal failure, may also occur

[1,4,5]. Several components have been isolated from Bothrops

venoms, including proteases such as serineproteinases and

metalloproteinases, phospholipase A2, L-amino acid oxidase, 59-

nucleotidase, hyaluronidase and C-type lectins [6], and these

comprise more than 90% of their dry weight [7]. Transcriptomic

analysis showed that 30% and 8% of the total transcripts present

in the venom gland of adult Bothrops jararaca encode metallo- and

serine-proteinases, respectively [8]. These enzymes have diversi-

fied amino acid sequences and display a variety of physiological

activities related to the pathogenesis of local and systemic reactions

[6,9,10].

The snake venom metalloproteinases (SVMPs) include enzymes

responsible for the cleavage of important tissue proteins such as

laminin, nidogen, fibronectin, collagen type IV, and proteoglycans

present in the endothelial basal membrane [9]. SVMPs, together

with ADAM (A disintegrin and metalloproteinase) enzymes, are

included in the M12B subfamily of zinc-dependent metalloprotei-

nases. SVMPs are multi-domain proteins that have been stratified

into three classes based on their domain composition: the mature

proteins of class P-I contain only a catalytic domain; the other

PLOS Neglected Tropical Diseases | www.plosntds.org 1 October 2013 | Volume 7 | Issue 10 | e2519

classes (P-II and P-III) contain additional non-catalytic domains,

such as disintegrin (or disintegrin-like), cysteine-rich and C-type

lectin-like domains [9].

The Complement (C) system is an integral part of the immune

system, protecting the host organism against invasion and

proliferation of various microorganisms. It is also involved in the

removal of the body’s own damaged and altered cells. The

Complement system consists of three activation pathways, i.e.,

classical, alternative and mannose binding lectin (MBL) pathways,

which merge at the proteolytic activation step of C3, a central

component of the system. Complement activation may generate

anaphylatoxins and membrane attack complex (MAC). Anaphyl-

atoxins (C3a, C4a, and C5a) are considered the bridge between

innate and adaptive immunity, and they are responsible for

controlling the local pro-inflammatory response through vasodi-

latation as well as chemotaxis and activation of leukocytes [11–15].

The regulatory mechanisms of Complement are extremely

balanced and are controlled by several Complement inhibitors,

such as membrane-bound Complement regulators and plasma

proteins such as C1-esterase inhibitor (C1-Inh), which regulate

activation of C1 and manose associated serine proteases (MASPs)

of the classical and MBL Complement pathways, among others

[11,16–18].

Previously, we analyzed the pro-inflammatory properties of

snake venoms from the genus Bothrops and demonstrated that

several of them were potent activators of the classical Complement

pathway [19]. This activation occurred in the absence of

sensitizing antibody and was, in part, associated with cleavage of

the C1-Inhibitor by metalloproteinases present in this venom,

resulting in disruption of Complement activation control. Some of

the Bothrops venom also activated the alternative and lectin

pathways. C3a, C4a and C5a were generated in sera treated with

the venom, not only through C-activation but also by direct

cleavage of Complement components [19].

The Complement system plays an important role in the defense

system, and also contributes to the amplification of inflammation if

activated in excess or inappropriately controlled. Thus, the aim of

the present study was to isolate and characterize a C-activating

protease from Bothrops pirajai venom to further understand the role

of Complement activation in the pathology of envenomation by

Bothrops snakes.

Materials and Methods

Ethics statementHuman blood was obtained from healthy donors who knew the

objectives of the study and signed the corresponding informed

consent form approved by the ethics committee (SISNEP

03689012.3.0000.5467).

All procedures involving animals were carried out in accordance

with ethical principles in animal research adopted by the Brazilian

Society of Animal Science and the National Brazilian Legislation

no. 11.794/08. The protocol was approved by the Institutional

Animal Care and Use Committee from the Butantan Institute

(permission no. 1109/13).

Chemicals, reagents and buffersBovine serum albumin (BSA), ortho-phenylenediamine (OPD),

1,10 phenanthroline (Phen), ethylene diamine tetraacetic acid

(EDTA), ethylene glycol bis-(b-aminoethyl ether)-N,N,N,N9-tetra-

cetic acid (EGTA), mannan, lipopolysaccharide (LPS) and human

IgM antibodies were purchased from Sigma (St. Louis, MI, USA).

Tween 20 and Phenylmethylsulfonyl fluoride (PMSF) were

purchased from Labsynth (Diadema, SP, Brazil) and Boehringer

Ingelheim (Ridgefield, CT, USA), respectively. Rabbit anti-goat

(RAG) and goat anti-rabbit (GAR) IgG labeled with horseradish

peroxidase (IgG-HRPO) were from Promega Corp. (Madison, WI,

USA). Rabbit IgG against C3 was from Santa Cruz Biotechnol-

ogy, Inc. (Santa Cruz, CA, USA). Purified human C3, C4, C5,

C1-Inh and goat anti-human C4 IgG were obtained from the

Quidell Corporation (San Diego, CA, USA). Fluorescent reso-

nance energy transfer (FRET) substrate, Abz-F-R-S-S-R-Q-

EDDnp (Abz-FRSSRQ-EDDnp), was synthesized and purified

according to Araujo et al. [20]. Cobra venom factor (CVF) was

purified in house from Naja naja venom as previously described

[21]. Mouse serum against rabbit erythrocytes was prepared in

house. The following buffers were used: Veronal-Buffered Saline

(VBS++), pH 7.4 (10 mM Na Barbitone, 0.15 mM CaCl2 and

0.5 mM MgCl2); BVB++ (VBS++, pH 7.2, containing 0.1% BSA);

alternative pathway (AP) buffer, pH 7.4 (5 mM Na-barbital,

10 mM EGTA, 7 mM MgCl2 and 150 mM NaCl); BAP (AP

buffer, pH 7.4, containing 0.1% BSA); BAP/Tween (BAP with

0.05% Tween 20); HEPES-buffered saline (HBS++), pH 7.4

(10 mM HEPES, 140 mM NaCl, 5 mM KCl, 1 mM CaCl2 and

1 mM MgCl2); phosphate-buffered saline (PBS), pH 7.2 (10 mM

Na Phosphate, 150 mM NaCl); and fluorescence activated cell

sorter (FACS) buffer (PBS, 1% BSA and 0?01% sodium azide).

VenomsBothrops pirajai and Naja naja venoms were supplied by the

Herpetology Laboratory from the Butantan Institute, Sao Paulo,

Brazil. Stock solutions were prepared in PBS buffer at 1.0 mg/mL.

The permission to access the venom of Bothrops pirajai (permission

no. 01/2009) was provided by the Brazilian Institute of Environ-

ment and Renewable Natural Resources (IBAMA), an enforce-

ment agency of the Brazilian Ministry of the Environment.

Protein purificationBothrops pirajai crude venom (50 mg) was applied to a FPLC-GP-

250 Plus system using a molecular exclusion column (Superose 12

10/300 GL, GE Life Sciences, USA), equilibrated with 0.05 M

ammonium bicarbonate, pH 7.8. Elution was carried out using the

Author Summary

The genus Bothrops inflicts the vast majority of snakebitesin Central and South America and is responsible for 90% ofsnake envenomations in Brazil. Envenomations are char-acterized by prominent local effects, including edema andnecrosis, and by systemic manifestations such as hemor-rhage, coagulopathy and acute renal failure. Severalcomponents have been isolated from Bothrops venoms,and the snake venom metalloproteinases (SVMPs) are keyenzymes contributing to the high toxicity of the venoms.Previously, we analyzed the pro-inflammatory propertiesof snake venoms from the genus Bothrops and demon-strated that several of them were potent activators of theComplement (C) system. C3a, C4a and C5a were generatedin venom-treated sera not only through C-activation butalso by direct cleavage of C-components. In the presentstudy, we have isolated and characterized a metallopro-teinase from Bothrops pirajai snake venom, named here asC-SVMP, which interferes with all three complementpathways, generating potent pro-inflammatory fragments,such as C3a, C4a and C5a. Our data suggest that C-activation by Bothrops pirajai venom is due to activity of anSVMP, which may play a role in the progression ofsymptoms that follow envenomation.

SVMP Activates the Complement System


same buffer at a flow rate of 30.0 mL/h. The active fraction

(screened for the ability to cleave C3) was chromatographed on a

Superdex 75 10/300 GL column (GE Life Sciences, USA), using the

same buffer and flow rate. Both fractionations were monitored by

ultraviolet absorption (280 nm). The molecular mass and homoge-

neity of the active fraction was assayed (w/v) by SDS–PAGE using a

12% polyacrylamide gel [22] and silver staining [23].

Mass spectrometry analysesThe protein digestion procedure was followed as proposed by

Kinter and Sherman [24] using 20 mg of the purified protein. In

the final procedure, the samples were dried using a speed-vac

(CHRIST, model RVC2-18, Osterode, Germany) and dissolved in

15 mL of formic acid 0.1% (v/v). An aliquot (4.5 mL) of the

resulting peptide mixture was separated by C18 column (75 mm

i.d.6100 mm) (Waters, Milford, MA) on an UPLC-ESI-Q-TOF

system (Waters, Milford, MA, USA) at a flow rate of 600 nL/min.

The gradient was 3–45% of solvent B (0.1% formic acid in

acetonitrile), 45–80% B in 2.5 min, hold at 80% B for 1 min, then

back to 97% of solvent A (0.1% formic acid in deionized water) in

1.5 min. The MS instrument was operated in data dependent

mode, in which one full MS scan was acquired in the m/z range of

200–2000 followed by MS/MS acquisition using collision-induced

dissociation of the 3 most intense ions from the MS scan. The

resulting fragment spectra were searched using the MASCOT

search engine (Matrix Science, UK) against the randomized

serpentes database (33870 sequences; 7677654 residues, down-

loaded from UniProt), with parent and fragment tolerances of

0.1 Da. Iodoacetamide derivatives of cysteine and methionine

oxidation were specified in MASCOT as variable modifications.

Only peptides that showed a significant threshold (p,0.05) in

MASCOT-based score were considered.

For the determination of molecular mass, the purified toxin

(200 ng) was acidified with formic acid 0.1% and analyzed by a Q-

ToF Ultima API spectrometer (Waters Corp.), operated in MS

continuum mode. Data were acquired from m/z 100–3.000 at a

scan rate of 1 s and an interscan delay of 0.1 s. The spectra were

accumulated over 30 scans, and the multiple charged data

produced on the m/z scale were converted to the mass scale

using the maximum entropy-based software [25] supplied with the

MassLynx 4.1 software package. The processing parameters were:

an output mass range of 22,000–24,000 Da at a resolution of

0.1 Da/channel; the simulated isotope pattern model was used

with the spectrum blur width parameter set to 0.2 Da; and the

minimum intensity ratios between successive peaks were 20% (left

and right).

Enzymatic activitySamples of purified venom protein (0.5 mg) were mixed with

5 mM Fluorescent Resonance Energy Transfer substrate and the

peptide Abz-FRSSRQ-EDDnp, in the presence or absence of

15 mM phenylmethylsulfonyl fluoride or 15 mM 1,10 phenan-

throline, which are inhibitors of serineproteinases and metallo-

proteinases, respectively. Reactions were carried out at 37uC using

a fluorescence spectrophotometer (lem = 420 nm and

lem = 320 nm; Victor 3TM, Perkin–Elmer, MA, USA) as described

by Araujo et al. [20]. All assays were performed in duplicate and

the specific proteolytic activity was expressed as micromoles of

cleaved substrate per minute per mg of protein (U/mg).

Normal human serumHuman blood samples were collected without anticoagulant and

allowed to clot for 2 hours at 4uC. After centrifugation, normal

human serum (NHS) was collected and stored at 280uC.

Treatment of the normal human serumFifty microliters of normal human serum (NHS) were incubated

with 50 mL of PBS buffer containing Bothrops pirajai crude venom

(1 mg) or purified venom protein (0.5 mg) for 30 min at 37uC. As a

negative control, NHS was incubated with PBS. After treatment,

the samples were tested for residual complement activity using the

enzyme-linked immunosorbant assay (ELISA).

Complement activation assaysMicrotiter plates were coated with 1 mg/well of IgM (Classical

Pathway), 1 mg/well of LPS (Alternative Pathway) or 10 mg/well

of mannan (Lectin Pathway) overnight at 4uC. The plates were

washed three times with PBS/0.05% Tween 20 and blocked with

1% BSA in PBS for 1 h at 37uC. After washing, serial dilutions of

normal human serum treated with PBS, Bothrops pirajai crude

venom or purified active protein were added. After incubation (1 h

at 37uC), the plates were washed with BVB+2/Tween (Classical

and Lectin Pathways) or BAP/Tween (Alternative Pathway)

Figure 1. Purification of the protease responsible for Complement activation. [A] Chromatogram of Bothrops pirajai venom (50 mg)fractionated on a FPLC-GP-250 Plus system using a molecular exclusion column (Superose 12 10/300 GL) in 0.05 M ammonium bicarbonate buffer,pH 7.8. [B] SDS-PAGE of fractions screened by the ability to cleave component C3. [C] Chromatogram of the fraction with activity (P2) on a Superdex75 10/300 GL column. [D] SDS-PAGE of fractions from Superdex 75 tested for the ability to cleave component C3. [E] SDS-PAGE followed by silverstaining to assess the purity of P3, the fraction capable of cleaving C3.doi:10.1371/journal.pntd.0002519.g001

Table 1. Mass spectrometry analysis of the purified protein.

PROTEIN ACCESSION NUMBER IDENTIFIED PEPTIDES (score 191)

DLLPR

ERDLLPR

Snake venom metalloproteinase P85314 YNSNLNTIR

BmooMPalfa-I KYNSNLNTIR

ETLKSFGEWR

HNPQCILNEPL

The primary sequence of the protein was identified by mass spectrometry.doi:10.1371/journal.pntd.0002519.t001



buffers and incubated with anti-human C4 (1:2.000; Classical and

Lectin Pathways) or anti-human C3 (1:5.000; Alternative Pathway)

for 1 h at 37uC. The plates were washed three times with BVB+2/

Tween or BAP/Tween and incubated for 1 h with the specific

anti-IgG antibody conjugated with HRPO. The plates were

washed and the reactions developed by adding OPD substrate

according to conditions established by the manufacturers (Sigma).

The absorbance was measured by an ELISA reader (Multiskan

spectrophotometer EX, Labsystems, Finland) at l492 nm. Normal

human serum arbitrarily set at 1000 aU/mL was used to generate

a calibration curve.

Direct cleavage of C-componentsThe proteolytic activity of B. pirajai crude venom or the purified

protease on C-components was assessed by SDS-PAGE under

reducing conditions. Briefly, 3 mg of each human purified C-

components, C3, C4, C5 or the C-regulatory protein C1-Inh, were

incubated with crude venom (1 mg), purified toxin (0.5 mg) or PBS

for 30 min at 37uC in the presence or absence of 15 mM 1,10-

phenanthroline. For the kinetic assays, increasing concentrations

of the purified toxin (0.01 mg–0.32 mg) were used. The cleavage of

the C-components was assessed using 10% (w/v) polyacrylamide

gels and silver staining. Using ImageJ 1.45 software (National

Institutes of Health, Bethesda, MD), the protein band density was

measured. The amount of protein under control conditions was

assigned a relative value of 100%. Alternatively, for amino-

terminal sequence analysis of the C3 protein fragments, gels were

subsequently electrotransferred to polyvinylidene difluoride

(PVDF) membranes and bands of interest were submitted to

Edman degradation using the PPSQ-10 protein sequencer

(Shimadzu, Tokyo, Japan).

Detection of anaphylatoxinsSamples of NHS (50 mL) or the human purified C-components

C3, C4 or C5 (3 mg) were incubated with crude venom (1 mg),

purified toxin (0.5 mg) or PBS for 30 min at 37uC, and C3a/C3a

desArg, C4a/C4a desArg and C5a/C5a desArg concentrations

were measured using the respective ELISA kit (BD Biosciences

PharMingen, CA, USA).

Figure 2. Proteolytic activity of C-SVMP upon FRET substrate.The proteolytic activity of C-SVMP (0.5 mg) on the FRET peptide (Abz-FRSSRQ-EDDnp - 5 mM in PBS) was determined in the presence orabsence of 15 mM of phenylmethylsulfonyl fluoride (PMSF) or 1,10phenanthroline (Phen). Reactions were carried out at 37uC using afluorescence spectrophotometer (lem = 420 nm and lem = 320 nm). Allassays were performed in duplicate and the specific proteolytic activitywas expressed as micromoles of cleaved substrate per minute per mg ofprotein (U/mg).doi:10.1371/journal.pntd.0002519.g002

Figure 3. Cleavage of purified human component C3. [A] Samples of purified human C3 (3 mg) were incubated with increasing concentrationsof C-SVMP (0.01–0.32 mg) or PBS for 30 min at 37uC. Cleavage was visualized by SDS-PAGE under reducing conditions followed by silver staining. [B]C3 is formed by two protein chains, b and a (1,641 amino-acid residues). The arrow indicates the site of cleavage for C-SVMP and the amino acidsdetermined by Edman degradation are underlined. Anaphylatoxin Domain (ANA).doi:10.1371/journal.pntd.0002519.g003



Analysis of the C1-Inhibitor regulatory activityThe MicroVue C1-lnhibitor Enzyme Immunoassay (Quidel,

San Diego, CA) was used to measure the amount of functional C1-

Inhibitor protein (3 mg) after treatment with increasing amounts of

purified toxin (0.5, 1, 2 mg) or PBS (control) in accordance with the

conditions established by the manufacturer.

Action of the purified toxin on the Complement systemin a murine model

Male Balb/c mice, aged 2 months and weighing 18–22 g, were

obtained from Central Animal Breeding from the Butantan

Institute, SP, Brazil. To measure the action of purified venom

protein on the complement system in vivo, BALB/c mice (groups of

3 animals) were injected i.p. with 10 mg of purified protein twice at

an interval of 24 h. As positive and negative controls, animals

received 10 mg of CVF or PBS buffer, respectively. Serum

complement activity was measured before treatment and 24 h

after the second injection. The results were representative of 3

independent assays.

Hemolytic assay for measurement of mouse serum C-activity

Blood was collected from mice and allowed to clot on ice. Sera

were separated and immediately assayed. Rabbit erythrocytes

were sensitized by incubation with mouse anti-rabbit erythrocyte

serum, washed and resuspended at 2% in VBS++. For each mouse

serum to be tested, doubling dilutions in VBS++ were made in the

wells of a 96-well plate (50 mL/well). Zero (VBS++) and 100% lysis

(H2O) controls were included. Antibody-sensitized rabbit erythro-

cytes (50 mL) were added to each well and incubated for 60 min at

37uC. The absorbance of the supernatant was measured at

415 nm and the percent hemolysis calculated by standard methods

[26].

Statistical analysisOne-way ANOVA, followed by the Bonferroni post-test, were

used to evaluate significant differences between control and

experimental results. Statistical analysis was performed using

GraphPad Prism software. Differences were considered statistically

significant when p values were p,0.05, p,0.01 or p,0.001.

Results

Purification and identification of the Bothrops C-activating protease

B. pirajai crude venom was fractionated using a Superose 12 10/

300 GL gel filtration column resulting in the elution of four

chromatographic peaks (Fig. 1A). All fractions were tested for the

ability to cleave purified human C3, as shown by a reduction in

size of the a-chain (Fig. 1B). A subset of proteins present in the

second chromatographic peak (Fig. 1A) were submitted to a

second gel filtration step using a Superdex 75 10/300 GL column.

The C3-cleaving activity was detected in Peak 3 (Fig. 1C, D),

which showed a single protein band of 23 kDa by SDS-PAGE

Figure 4. Action of C-SVMP on complement pathways. Samples (50 ml) of normal human serum (NHS), as the complement source, wereincubated for 30 min at 37uC with 50 ml of C-SVMP (0.5 mg) and with 50 ml of Bothrops venom (1 mg) or PBS as positive and negative controls,respectively. The residual complement activity was determined by ELISA for the Classical [A], Alternative [B] and Lectin [C] pathways. The results wereexpressed as the percentage reduction of component deposition relative to the negative control sample (NHS+PBS). Generation of anaphylatoxins inthe serum samples was measured using ELISA kits for C3a [D], C4a [E] and C5a [F]. The results were expressed as the concentration of eachanaphylatoxin per mL of human serum. Data are representative of three separate experiments and are expressed as the mean of the duplicates +/2SD. *p,0.05; **p,0.01; ***p,0.001.doi:10.1371/journal.pntd.0002519.g004



(Fig. 1E). Analysis by ESI-MS of intact purified protein confirmed

its molecular mass as being 23145.6543 Da (Fig. S1). Analysis of

the same protein by in solution trypsin digestion and LC-MS/MS

resulted in the identification of six tryptic peptides that matched

sequences of the P-I class of SVMPs from Bothrops venom (Table 1).

The proteolytic activity of the purified protein, here named as

C-SVMP, was tested using the FRET substrate Abz-FRSSRQ-

EDDnp, which contains a universal sequence recognized by

proteases of different catalytic natures. C-SVMP cleaved the

FRET substrate with high activity (Fig. 2). To assess and confirm

the enzymatic nature of the C-SVMP, the assay was performed in

the presence of 1,10-phenanthroline or phenylmethylsulfonyl

fluoride, which are inhibitors of metallo- and serine-proteinases,

respectively. The proteolytic activity of C-SVMP was completely

inhibited by Phen, while PMSF had no effect, identifying C-

SVMP as a metalloproteinase.

C-SVMP cleaves the a-chain of C3 in a dose dependentmanner to generate biologically active C3a

The action of purified C-SVMP on C3 was assessed by SDS-

PAGE. C-SVMP dose-dependently hydrolyzed the alpha chain of

purified human C3, resulting in a slightly lower Mr, while the beta

chain was not affected (Fig. 3A). Analysis by Edman degradation

of the cleaved C3 alpha chain revealed the N-terminal amino acid

sequence Ser-Asn-Leu-Asp-Glu, suggesting that C-SVMP cleaves

the C3 alpha chain at the same site as C3 convertases after

Complement activation, thus generating C3b and C3a (Fig. 3B).

C-SVMP interferes with the classical, alternative and lectincomplement pathways

We previously showed that crude Bothrops spp. venoms

interfered with all three Complement activation pathways [19].

Figure 5. Proteolytic activity of C-SVMP on human purified Complement components. The C-components, C3, C4 and C5 (3 mg) wereincubated with increasing concentrations of C-SVMP (0.01 mg–0.32 mg). Cleavage of C-components was visualized by SDS-PAGE (10%) underreducing conditions followed by silver staining. The percentage cleavage of the a chain (C3, C4 and C5) was quantified by densitometry [A–C].Purified human complement proteins C3, C4 and C5 (3 mg) were also incubated with purified C-SVMP (0.5 mg) or Bothrops venom (1 mg) in thepresence or absence of 1,10 phenanthroline (Phe - 15 mM), a metalloproteinase inhibitor [D–F]. Generation of anaphylatoxins, after treatment of C3,C4 or C5 samples (3 mg) with C-SVMP (0.5 mg) or Bothrops venom (1 mg), was determined by ELISA [G–I]. Data are representative of three separateexperiments. **p,0.01; ***p,0.001.doi:10.1371/journal.pntd.0002519.g005



Figure 6. Proteolytic activity of C-SVMP on C1-Inh and analysis of its regulatory activity. [A] Human purified C1-Inh samples (3 mg) wereincubated with purified C-SVMP (0.5 mg) or Bothrops venom (1 mg) in the presence or absence of 1,10 phenanthroline (Phe - 15 mM). [B] Alternatively,samples of purified human C1-Inh (1 mg) were incubated with increasing concentrations of C-SVMP (0.5 mg, 1 mg and 2 mg). The cleavage wasvisualized by SDS-PAGE (10%) under reducing conditions followed by silver staining. [C] C1-Inh activity was determined using the MicroVue ELISA kit(***p,0.001). Data are representative of two separate experiments.doi:10.1371/journal.pntd.0002519.g006



To assess whether C-SVMP could also affect the Complement

activation pathways, a microtiter plate-based assay was used.

Human serum was pre-incubated with C-SVMP and the

remaining Complement activity was measured. Similar to crude

venom, C-SVMP induced a reduction in the deposition of

Complement components (C4b, as determined for the classical

and lectin pathways – Figs. 4 A and B; C3b for the alternative

pathway - Fig. 4C), demonstrating that pre-incubation with C-

SVMP resulted in Complement activation and consumption. The

consumption of the classical pathway was approximately 2-fold

greater than that of the alternative and lectin pathways.

Reduction in C4b and C3b deposition could also have resulted

from direct inhibition, rather than from activation and consump-

tion. The activating rather than inhibitory action of C-SVMP on the

complement system cascade was confirmed by detection of three

anaphylatoxins, i.e., C3a, C4a and C5a (Figure 4D–E). The

generation of C3a and C4a after C-SVMP treatment was

approximately 3-fold higher than the untreated sera; and C5a was

approximately 6-fold higher. Interestingly, C5a generation induced

by C-SVMP was greater than that induced by whole venom, while

C3a and C4a generation induced by C-SVMP was less.

C-SVMP directly cleaves C4, C5 and the C1-InhibitorWe have previously shown that crude Bothrops spp. venoms

directly cleaved not only C3, which was used here as a guide

during the isolation of C-SVMP, but also complement compo-

nents C4, C5 and the inhibitor of the classical and lectin pathways,

the C1-Inhibitor [19]; therefore, the possible direct cleavage of

these components by C-SVMP was assessed by SDS-PAGE and

densitometry. Figure 5 (A–C) shows that C-SVMP incubation with

C3, C4 and C5 resulted in a dose dependent hydrolysis of the

alpha chains of these C-components, as shown by a reduced Mr of

the alpha chains (Fig. 5 D–F); the Mr of the beta and gamma (C4

only) chains were not affected. Concomitantly with the reduction

in Mr of the alpha chains, C3a, C4a and C5a fragments, as

detected by specific ELISA, were generated (Fig. 5 G–I).

Cleavage of the alpha chains of C3, C4 and C5 by C-SVMP

was completely inhibited by 1,10-phenanthroline; however, this

compound only partially inhibited the action of venom on these

proteins (Fig. 5 D–F), suggesting that crude venom, in addition to

the metalloproteinase C-SVMP isolated here, contains another

protease, most likely a serineproteinase.

The cleavage of C1-Inh was completely inhibited by 1,10-

phenanthroline after treatment with either C-SVMP or crude

venom (Fig. 6A), suggesting that C-SVMP may be solely

responsible for cleavage of C1-Inh. Moreover, C-SVMP also dose

dependently cleaved C1-Inh, generating a fragment of ,83 kDa,

which was positively associated with the dose dependent reduction

in the level of the functionally active C1-Inhibitor protein

(Fig. 6B,C).

C-SVMP is also able to activate the complement systemin vivo

Because C-SVMP is able to activate human complement, as

determined in in vitro assays, we analyzed its action in vivo using a

murine model. As a positive control, mice were treated with CVF,

Figure 7. The complement activation property of C-SVMP measured in vivo. BALB/c mice (n = 3) were twice injected i.p. with C-SVMP(10 mg/dose) at intervals of 24 h. As positive and negative controls, animals received CVF (10 mg/dose) or PBS, respectively. Serum samples werecollected immediately prior to starting treatment and at 24 h after the last injection. Complement activity in the individual serum samples wasmeasured and expressed as the percentage of hemolysis. The results were representative of three independent assays. *p,0.05; **p,0.01;***p,0.001.doi:10.1371/journal.pntd.0002519.g007



a well-known complement activating molecule isolated from the

venom of Naja naja. Figure 7 shows that C-SVMP induced partial

depletion of the complement system in vivo, as determined by the

significantly reduced hemolytic activity in sera from treated mice

compared to the sera of untreated animals. Under the conditions

used, C-SVMP was less effective in consuming complement than

CVF.

Discussion

Complement activation by an animal’s toxin and its role in the

envenomation remains poorly studied, but has been described for

some snake venoms belonging to the Elapidae and Viperidae families

[27–33,19].

The best-described complement activating protein isolated from

snake venom is CVF, a C3-like molecule that complexes with

factor B, which is cleaved and activated by factor D, generating a

very stable C3/C5-convertase that leads to excessive complement

activation and depletion [27]. There is no evidence that CVF has

enzymatic activity upon individual components of the complement

system, and it can only indirectly trigger the activation of the

system.

In this study we have isolated and characterized a metallopro-

teinase from Bothrops snake venom, C-SVMP, that interferes with

the complement system. SVMPs are known for their key role in

Bothrops snake envenomation, producing prominent local tissue

damage characterized by edema, pain, hemorrhage, dermone-

crosis and inflammatory reaction, as well as by systemic effects,

such as coagulopathies, nephrotoxicity, hemodynamic dysfunction

and cardiotoxicity [34–37].

C-SVMP has a single polypeptide chain with a molecular mass

of 23.145 kDa (Fig. 1 and S1) and exhibits high homology with P-I

class zinc metalloproteinases from Bothrops venom (Table 1), i.e.,

BmooMPalfa-I from B. moojeni (accession number: P85314) [38],

based on a partial sequence obtained by trypsin digestion and mass

spectrometry. Recently, it was isolated from the B. pirajai venom a

new P1-class metalloproteinase with fibrin(ogen)olytic and throm-

bolytic activities [39], whose identity with the C-SVMP still needs

further analysis, once the internal sequences obtained are similar

to others P-I SVMP from Bothops spp. Despite the high similarity

between those SVMP, they might act in specific substrates.

The metalloproteinase activity of C-SVMP was confirmed by

cleavage of the FRET substrate peptide Abz-FRSSRQ-EDDnp

(Fig. 2), which was inhibited by 1,10-phenanthroline but not by

phenylmethylsulfonyl fluoride, thereby confirming that C-SVMP

is a metalloproteinase.

Members of the P-I class of SVMPs, the small SVMPs, have

molecular masses of 20–25 kDa. The catalytic domain contains a

zinc-binding sequence, HEXXHXXGXXH, plus a Met-Turn

sequence, CI/VM, which stabilizes the three Histidine residues

important for enzymatic catalysis of the substrates; the zinc

binding region is strictly conserved among the SVMPs [9].

Numerous biological activities are attributed to P-I class SVMPs

in the context of the envenoming pathology, such as fibrinolysis

and fibrinogenolysis, hemorrhage, myonecrosis, inflammation,

apoptosis and prothrombin activation in addition to inhibition of

platelet aggregation activities [34–39]. However, the effect on the

complement system has not been investigated.

Our current data show that C-SVMP, isolated based on its C3-

cleaving ability, interferes with all three complement pathways but

preferentially interferes with the classical pathway. C-SVMP

generated bioactive fragments, such as C3a, C4a and C5a, as

confirmed by ELISA (Fig. 4), and C3b, as determined by the

amino acid sequence analysis of the cleaved N-terminal C3-alpha

chain, Ser-Asn-Leu-Asp-Glu (Fig. 3). This cleavage site is identical

to that accomplished following complement activation via any of

the three activation pathways [40].

Interestingly, Sun and Bao [41] have isolated from Naja atra

venom a PIII metalloproteinase (termed atrase B) with anticom-

plementary activity. This protease shows fibrinogenolytic activity

and edema-inducing activity, but has no hemorrhagic activity.

Atrase B inhibits the hemolytic activity of the complement system

by cleaving C-components C6, C7, C8 and factor B, thus affecting

MAC assembly. Moreover, differently from C-SVMP from B.

pirajai, atrase B has no enzymatic activity on C1, C2, C3, C4 and

C5.

In addition to cleaving and activating C3, C4 and C5 (Figs. 4

and 5), C-SVMP also directly cleaved C1-Inh (Fig. 6), a regulatory

protein that binds and inactivates C1r, C1s, MASP-1 and MASP-

2, as well as interfering with the regulation of the coagulation

cascade. C1r and C1s activate the classical complement pathway,

while MASP-1 and MASP-2 activate the lectin pathway [42].

Cleavage of C1-INH by Crotalid, Viperid and Colubrid snake

venoms has previously been reported by Kress et al. [43], who

demonstrated that the inhibitor was converted into an active 89-

kDa intermediate species and then further cleaved to form an 86-

kDa inactive inhibitor. Inactivation, even partial, of the C1-

Inhibitor by C-SVMP action (Fig. 6) may lead to auto-activation of

the classical and lectin pathways [18,44]. The more potent action

of C-SVMP on the classical pathway compared to the lectin

pathway may possibly be explained by stronger inhibition of the

classical pathway by C1-Inh, although this remains to be

investigated. Inactivation of C1-Inh not only results in uncon-

trolled C-derived anaphylatoxin generation but also uncontrolled

bradykinin generation [18,44]; all of these products contribute to

vasodilatation and increased spreading of the venom.

In vivo, C-SVMP induced consumption of murine complement

components most likely via activation of the classical pathway

and/or direct cleavage of C3, thereby reducing lytic activity of the

sera (Fig. 7). The reduction of serum lytic activity was not as great

as the reduction observed following injection of CVF, showing that

CVF is more efficient in dysregulating the complement system.

This may be explained by the different dysregulation mechanisms

of the complement system; while C-SVMP causes dysregulation by

direct enzymatic cleavage, CVF forms a very stable C3/C5

convertase with factor B, which has an in vivo half-life of 11.5 h

[45] and thus can cause prolonged C-activation. Furthermore, due

to its smaller size (Mw 23 kDa), C-SVMP may get cleared from

circulation more easily than the much larger CVF (Mw 150 kDa,

which increases to over 200 kDa when complexed with fB, thereby

extending the in vivo half-life).

In conclusion, we show here that a P-I metalloproteinase from

Bothrops pirajai snake venom is able to activate the complement

system by direct cleavage of central C-components, i.e., C3, C4

and C5, thereby generating biologically active fragments, such as

anaphylatoxins, and by cleaving the C1-Inhibitor, which may

affect Complement activation control. These results suggest that

direct complement activation by SVMPs may play a role in the

progression of symptoms following envenomation.

Supporting Information

Figure S1 Molecular mass determination of C-SVMP (300 ng)

was performed by electrospray ionization (ESI) coupled with mass

spectrometry using a Q-ToF instrument.

(TIF)



Acknowledgments

We thank Dr. Isaura Y. Hirata, Departmento de Biofısica, Escola Paulista

de Medicina, Universidade Federal de Sao Paulo, for her support with the

Edman degradation analysis.

Author Contributions

Conceived and designed the experiments: GPQ DVT. Performed the

experiments: GPQ FCM ASL. Analyzed the data: GPQ FCVP SMTS

ASL AFPL CWvdB DVT. Contributed reagents/materials/analysis tools:

FCVP SMTS AFPL DVT. Wrote the paper: GPQ FCVP SMTS ASL

AFPL CWvdB DVT.

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p-i snake venom metalloproteinase is able to activate the

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